SARS-CoV受体ACE-2基因的克隆及在真核细胞中的表达

目的克隆、真核表达严重急性呼吸综合征冠状病毒(SARS-CoV)受体ACE2基因,建立安全、可靠的SARS-CoV保护性体液免疫评价体系。方法采用Trizol一步法提取人右心衰心房组织总RNA,逆转录巢式聚合酶链反应(RT-nested-PCR)扩增ACE2全长基因,克隆人pcDNA4/HisMax-TOPO表达载体,重组质粒转染293T细胞进行瞬时表达,Western Blot检测其真核表达;建立细胞融合抑制实验以检测SARS-CoV中和抗体,并与SARS-CoV假病毒中和试验进行平行比较。结果重组质粒可在真核细胞中表达ACE2蛋白,其蛋白表达细胞可与S蛋白表达细胞产生细胞融合现象,并可用于中和抗体的检测;其结果与SARS-CoV假病毒中和试验较为一致。结论克隆和真核表达了SARS-CoV受体ACE-2基因;基于其蛋白真核表达的细胞融合抑制实验可用于SARS-CoV中和抗体的检测。

Cloning of ACE-2 gene encoding the functional receptor for the SARS coronavirus and its expression in eukaryotic cells

Objective Angiotensin-converting enzyme 2(ACE-2) has been identified as a functional receptor of severe acute respiratory syndrome coronavirus(SARS-CoV),so its gene was cloned and eukaryotic expressed for further insight into mechanisms in SARS-CoV entry and pathogenesis,as well as development of a safe and reliable neutralization assay for SARS-CoV. Methods Total RNA was extracted from right atrial tissue of a patient with right heart failure resected during a valvular replacement surgery by Trizol one-step method,and the full-length ACE-2 encoding gene was acquired by RT-nested-PCR. The ACE-2 encoding gene was then cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to construct the recombinant plasmid pcDNA4/ ACE-2,which was then transfected into 293 T cell and ACE-2 eukaryotic transient expression was detected by Western Blot. Syncytia inhibition assay was established to detect SARS-CoV neutralizing antibody, and compared parallelly with SARS pseudovirus neutralization assay. Results The recombinant plasmid pcDNA4/ ACE-2 could express ACE-2 protein in eukaryotic cells and induce cell-cell fusion between S protein- and ACE2-expressing cells.This cell-cell fusion assay could be used to detect SARS-CoV neutralizing antibody. Conclusion SARS-CoV receptor ACE-2 gene was successfully cloned and eukaryotic expressed, and used to establish syncytia inhibition assay for SARS-CoV neutralizing antibody assay.